A comparison of mitochondrial DNA isolation methods in frozen post-mortem human brain tissue--applications for studies of mitochondrial genetics in brain disorders

Given that many brain disorders are characterized by mitochondrial dysfunction, there is a growing interest in investigating genetic and epigenetic variation in mitochondrial DNA (mtDNA). One major caveat for such studies is the presence of nuclear-mitochondrial pseudogenes (NUMTs), which are regions of the mitochondrial genome that have been inserted into the nuclear genome over evolution and, if not accounted for, can confound genetic studies of mtDNA. Here we provide the first systematic comparison of methods for isolating mtDNA from frozen post-mortem human brain tissue. Our data show that a commercial method from Miltenyi Biotec, which magnetically isolates mitochondria using antibodies raised against the mitochondrial import receptor subunit TOM22, gives significant mtDNA enrichment and should be considered the method of choice for mtDNA studies in frozen brain tissue.Alzheimer's Research U

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.This article is freely available via Open Access. Click on the publisher URL to access it via the publisher's site.We acknowledge funding from Wellcome ( 200990 ). S.E. is a Wellcome Senior Investigator. U.F.P. is supported by a predoctoral fellowship from the Basque Government ( PRE_2018_1_0253 ). M.M.O. is supported by a predoctoral fellowship from the University of the Basque Country ( UPV/EHU, PIF 2018 ). I.J.H. is supported by the Carlos III Health Program ( PI17/00380 ), and País Vasco Department of Health ( 2018111043 ; 2018222031 ). A.S. is supported by the UK Medical Research Council with a Senior Non-Clinical Fellowship ( MC_PC_13029 ). T. Harel is supported by the Israel Science Foundation grant 1663/17 . W.H.Y. is supported by the National Institute of General Medical Sciences of the National Institutes of Health through grant 5 P20 GM103636-07 . J.R.L. is supported by the US National Institute of Neurological Disorders and Stroke ( R35NS105078 ), the National Institute of General Medical Sciences ( R01GM106373 ), and the National Human Genome Research Institute and National Heart Lung and Blood Institute (NHGRI/NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542 ). R.W.T. is supported by the Wellcome Centre for Mitochondrial Research ( 203105/Z/16/Z ), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease , Mitochondrial Disease Patient Cohort (UK) ( G0800674 ), the UK NIHR Biomedical Research Centre for Aging and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust, the MRC/EPSRC Molecular Pathology Node , The Lily Foundation , and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children . The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). This study makes use of DECIPHER, which is funded by Wellcome. See Nature PMID: 25533962 or https://www.ddduk.org/access.html for full acknowledgment.pre-print, post-print (6 month embargo

NASAwide electronic publishing system: Prototype STI electronic document distribution, stage-4 evaluation report

This evaluation report contains an introduction, seven chapters, and five appendices. The Introduction describes the purpose, conceptual frame work, functional description, and technical report server of the STI Electronic Document Distribution (EDD) project. Chapter 1 documents the results of the prototype STI EDD in actual operation. Chapter 2 documents each NASA center's post processing publication processes. Chapter 3 documents each center's STI software, hardware, and communications configurations. Chapter 7 documents STI EDD policy, practices, and procedures. The appendices, which arc contained in Part 2 of this document, consist of (1) STI EDD Project Plan, (2) Team members, (3) Phasing Schedules, (4) Accessing On-line Reports, and (5) Creating an HTML File and Setting Up an xTRS. In summary, Stage 4 of the NASAwide Electronic Publishing System is the final phase of its implementation through the prototyping and gradual integration of each NASA center's electronic printing systems, desktop publishing systems, and technical report servers to be able to provide to NASA's engineers, researchers, scientists, and external users the widest practicable and appropriate dissemination of information concerning its activities and the result thereof to their work stations

Convergence of the Optimized Delta Expansion for the Connected Vacuum Amplitude -- Anharmonic Oscillator

The convergence of the linear $\delta$ expansion for the connected generating functional of the quantum anharmonic oscillator is proved. Using an order-dependent scaling for the variational parameter $\lambda$, we show that the expansion converges to the exact result with an error proportional to $\exp(-cN^{1/3})$.Comment: LaTeX, 14 pages, 4 figures

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease

The Impact of Global Warming and Anoxia on Marine Benthic Community Dynamics: an Example from the Toarcian (Early Jurassic)

The Pliensbachian-Toarcian (Early Jurassic) fossil record is an archive of natural data of benthic community response to global warming and marine long-term hypoxia and anoxia. In the early Toarcian mean temperatures increased by the same order of magnitude as that predicted for the near future; laminated, organic-rich, black shales were deposited in many shallow water epicontinental basins; and a biotic crisis occurred in the marine realm, with the extinction of approximately 5% of families and 26% of genera. High-resolution quantitative abundance data of benthic invertebrates were collected from the Cleveland Basin (North Yorkshire, UK), and analysed with multivariate statistical methods to detect how the fauna responded to environmental changes during the early Toarcian. Twelve biofacies were identified. Their changes through time closely resemble the pattern of faunal degradation and recovery observed in modern habitats affected by anoxia. All four successional stages of community structure recorded in modern studies are recognised in the fossil data (i.e. Stage III: climax; II: transitional; I: pioneer; 0: highly disturbed). Two main faunal turnover events occurred: (i) at the onset of anoxia, with the extinction of most benthic species and the survival of a few adapted to thrive in low-oxygen conditions (Stages I to 0) and (ii) in the recovery, when newly evolved species colonized the re-oxygenated soft sediments and the path of recovery did not retrace of pattern of ecological degradation (Stages I to II). The ordination of samples coupled with sedimentological and palaeotemperature proxy data indicate that the onset of anoxia and the extinction horizon coincide with both a rise in temperature and sea level. Our study of how faunal associations co-vary with long and short term sea level and temperature changes has implications for predicting the long-term effects of “dead zones” in modern oceans

The HNF4A R76W mutation causes atypical dominant Fanconi syndrome in addition to a β cell phenotype

types: JOURNAL ARTICLEMutation specific effects in monogenic disorders are rare. We describe atypical Fanconi syndrome caused by a specific heterozygous mutation in HNF4A. Heterozygous HNF4A mutations cause a beta cell phenotype of neonatal hyperinsulinism with macrosomia and young onset diabetes. Autosomal dominant idiopathic Fanconi syndrome (a renal proximal tubulopathy) is described but no genetic cause has been defined.This article presents independent research supported by the National Institute for Health Research (NIHR) Exeter Clinical Research Facility. The research is funded by a Wellcome Trust Senior Investigator Award, (grant number 098395/Z/12/Z).Wellcome Trus

Milestones in the Observations of Cosmic Magnetic Fields

Magnetic fields are observed everywhere in the universe. In this review, we concentrate on the observational aspects of the magnetic fields of Galactic and extragalactic objects. Readers can follow the milestones in the observations of cosmic magnetic fields obtained from the most important tracers of magnetic fields, namely, the star-light polarization, the Zeeman effect, the rotation measures (RMs, hereafter) of extragalactic radio sources, the pulsar RMs, radio polarization observations, as well as the newly implemented sub-mm and mm polarization capabilities. (Another long paragraph is omitted due to the limited space here)Comment: Invited Review (ChJA&A); 32 pages. Sorry if your significant contributions in this area were not mentioned. Published pdf & ps files (with high quality figures) now availble at http://www.chjaa.org/2002_2_4.ht

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.IB is funded by Wellcome (WT206194). ATH and SE are the recipients of a Wellcome Trust Senior Investigator award and ATH is employed as a core member of staff within the NIHR funded Exeter Clinical Research Facility and is an NIHR senior investigator. EDF was a Naomi Berrie Fellow in Diabetes Research during the study. SEF has a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 105636/Z/14/Z). CCW holds a Wellcome Trust Intermediate Clinical Fellowship (Grant Number: 105914/Z/14/Z). HH is funded by the Research Foundation-Flanders (FWO), the VUB Research Council and Stichting Diabetes Onderzoek Nederland

Designing an intervention to help people with colorectal adenomas reduce their intake of red and processed meat and increase their levels of physical activity: a qualitative study

Background: most cases of colorectal cancer (CRC) arise from adenomatous polyps and malignant potential is greatest in high risk adenomas. There is convincing observational evidence that red and processed meat increase the risk of CRC and that higher levels of physical activity reduce the risk. However, no definitive randomised trial has demonstrated the benefit of behaviour change on reducing polyp recurrence and no consistent advice is currently offered to minimise patient risk. This qualitative study aimed to assess patients' preferences for dietary and physical activity interventions and ensure their appropriate and acceptable delivery to inform a feasibility trial.Methods: patients aged 60-74 included in the National Health Service Bowel Cancer Screening Programme (NHSBCSP) were selected from a patient tracking database. After a positive faecal occult blood test (FOBt), all had been diagnosed with an intermediate or high risk adenoma (I/HRA) at colonoscopy between April 2008 and April 2010. Interested patients and their partners were invited to attend a focus group or interview in July 2010. A topic guide, informed by the objectives of the study, was used. A thematic analysis was conducted in which transcripts were examined to ensure that all occurrences of each theme had been accounted for and compared.Results: two main themes emerged from the focus groups: a) experiences of having polyps and b) changing behaviour. Participants had not associated polyp removal with colorectal cancer and most did not remember being given any information or advice relating to this at the time. Heterogeneity of existing diet and physical activity levels was noted. There was a lack of readiness to change behaviour in many people in the target population.Conclusion: this study has demonstrated the difficulties involved in developing interventions to change dietary and physical activity behaviour in this population. The need to tailor the intervention to individuals, the lack of knowledge about the aetiology of colon cancer and the lack of motivation to change behaviour are critical factors